Mar 30, 2023

Public workspaceSingle cell dissociation of brain organoids

DOI
dx.doi.org/10.17504/protocols.io.e6nvwj799lmk/v1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Federico Bertoli
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwj799lmk/v1
Protocol Citation: michela.deleidi, María José Pérez J., Hariam Raji 2023. Single cell dissociation of brain organoids. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj799lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79538
Keywords: brain organoids, cell dissociation, Papain Dissociation System, ASAPCRN
Abstract
This protocol details about single cell dissociation of brain organoids.
Attachments
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Materials
Kit:
Papain Dissociation System. ReagentPapain Dissociation SystemWorthington Biochemical CorporationCatalog #LK003150

Reconstitute powders.
  • Add Amount5 mL Earle´s medium into Papain Vial (1 Vial/2 organoids).
  • Add Amount500 µL Earles´s medium into DNAse vial.
  • Add Amount35 mL Earle´s medium into Inhibitor vial (1 vial/10 organoids).



Single cell dissociation of brain organoids
Single cell dissociation of brain organoids
27m
27m
Mix Amount500 µL DNAse with Amount5 mL Papain.
Note
Note: MIX GENTLY.



Mix
Transfer single or pooled organoid to 60 mm dish.
Aspirate excess media, add Amount2.5 mL Papain + DNAse solution.

Pipetting
With a razor blade mince organoid (<1 mm).
Transfer plate to an orbital shaker Shaker70 rpm, 00:30:00 (inside incubator).

With 1-mL pipette dissociate pieces (Mix up-down 30 times).
Put in orbital shaker Duration00:20:00 .

20m
In the meantime, add Amount5 mL Earle´s medium + Amount3 mL Inhibitor to a 15-mL conical tube.

Pipetting
Remove samples from the orbital shaker. With a 1-mL tip, mix up-down 30 times.
Mix
Take Amount2 mL (upper part) into new tube using a 40 µm cell strainer. Wait 1-3 min to debris to settle.

Transfer cell suspension to the inhibitor tube. Invert to mix 5 times.
Mix
Centrifuge Centrifigation300 rpm, Room temperature, 00:07:00 .

7m
Centrifigation
Aspirate supernatant, resuspend in Amount500 µL to Amount1 mL 0.5% BSA-PBS (Up-down 30 times).

Filter the resuspended cells (Amount900 µL ) with a 30 µm cell strainer.

Count the cells for the final suspension and dilute. Resuspend at 1000 cells/μl in 0.04% BSA-PBS.